[unreadable] P-selectin, expressed on activated endothelial cells and platelets, is a cell surface glycoprotein which mediates host cell-tumor cell interactions relevant to the process of blood-borne metastasis. The most convincing evidence for a direct role of P-selectin in hematogenous spread is the marked inhibition of metastasis in P-selectin-deficient mice compared to wild-type controls. Thus, identification of "functional" ligands on human tumor cells responsible for P-selectin-mediated host-tumor cell adhesion will be important to the understading of hematogenous metastasis and may offer novel avenues for its control. Although P-selectin ligands have been identified on tumor cell lines, their function and biologic significance have yet to be demonstrated in a dynamic (non-static) setting. As has been argued in the literature, distinctions must be drawn between structures that can bind to selectins under static conditions in vitro, and "functional" ligands that do interact with selectins under flow conditions in vivo. Moreover, the detailed biophysical characteristics of P-selectin binding to P-selectin ligands on tumor cells have not been previously explored. In this application, a human colon carcinoma cell model (LS174T) is used for the identification, isolation, and biochemical and biophysical characterization of the P-selectin ligand(s), since colon cancer is prevalent in the United States and is also among those tumors with a propensity for hematogenous spread. Aim 1 tests the hypothesis that the P-selectin ligand on intact LS174T cells is a novel protease-sensitive, O-linked sialylated glycoconjugate, using flow-based adhesion assays and highly specific enzymes, antibodies and glycoconjugate biosynthesis inhibitors. Moreover, the dissociation rate constant and strength of P-selectin- ligand bonds between P-selectin and intact LS174T cells will be determined using a molecular force probe. In Aim 2, we will isolate the putative P-selectin ligand(s) from LS174T cells using affinity chromatography coupled with standard molecular and cell biology techniques. Aim 3 will characterize the biochemical and biophysical properties of the isolated ligand(s), and test its ability to support P-selectin-mediated binding under flow, as well as its cross-reactivity with L- and E- selectin. Knowledge gained from these studies will advance the design of novel anti-metastatic agents, and our understanding of the roles of P-selectin/ligand interactions in metastasis. [unreadable] [unreadable]